Methods for prognosis and monitoring cancer therapy

ABSTRACT

The present invention also relates to biomarkers and the use of biomarkers for the prediction and prognosis of cancer as well as the use of biomarkers to monitor the efficacy of cancer treatment. Specifically, this invention relates to the use of HER-2, EGFR, VEGF, u-PA, p-PAI-1, and soluble forms thereof, as biomarkers for cancer, especially for subjects treated with sorafenib.

This application claims the benefit of the filing date of U.S.Provisional Application Ser. No. 60/731,278 filed Oct. 31, 2005, theentire disclosure of which is incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to biomarkers and the use of biomarkersfor the prediction and prognosis of cancer as well as the use ofbiomarkers to monitor the efficacy of cancer treatment. Specifically,this invention relates to the use of HER-2, EGFR, VEGF, u-PA(urokinase-plasminogen activator), p-PAI-1; and soluble forms thereof,as biomarkers as biomarkers for cancer.

BACKGROUND OF THE INVENTION

Many disease states are characterized by differences in the expressionlevels of various genes either through changes in the copy number of thegenetic DNA or through changes in levels of transcription of particulargenes (e.g., through control of initiation, provision of RNA precursors,RNA processing, etc.). For example, losses and gains of genetic materialplay an important role in malignant transformation and progression.These gains and losses are thought to be driven by at least two kinds ofgenes, oncogenes and tumor suppressor genes. Oncogenes are positiveregulators of tumorgenesis, while tumor suppressor genes are negativeregulators of tumorgenesis (Marshall, Cell 64:313-326, 1991; Weinberg,Science 254:1138-1146, 1991). Therefore, one mechanism of activatingunregulated growth is to increase the number of genes coding foroncogene proteins or to increase the level of expression of theseoncogenes (e.g., in response to cellular or environmental changes), andanother mechanism is to lose genetic material or to decrease the levelof expression of genes that code for tumor suppressors. This model issupported by the losses and gains of genetic material associated withglioma progression (Mikkelson, et al., J. Cellular Biochem. 46:3-8,1991). Thus, changes in the expression (transcription) levels ofparticular genes (e.g., oncogenes or tumor suppressors) serve assignposts for the presence and progression of various cancers.

DESCRIPTION OF THE INVENTION

The present invention relates to biomarkers and the use of biomarkersfor the prediction and prognosis of cancer as well as the use ofbiomarkers to monitor the efficacy of cancer treatment. Specifically,this invention relates to the use of HER-2, EGFR, VEGF, u-PA(urokinase-plasminogen activator), p-PAI-1, and soluble forms thereof,as biomarkers for cancer (including both solid tumors, metastatictumors, and blood cancers), such as breast cancer, colon carcinoma,melanoma, renal cell carcinoma, non-small cell lung cancer, acutemyeloid leukemia, and myelodyspastic syndrome, and hepatocellularcancer, especially for subjects treated with sorafenib and otherdiarylureas.

In addition, it is an objective of the invention to provide methods andreagents for the prediction, diagnosis, prognosis, and therapy ofcancer.

In one embodiment of the present invention, the biomarkers comprise oneor more genes and/or gene products that demonstrate altered expressionfollowing exposure to sorafenib and other diarylureas.

Sorafenib is the tosylate salt of4-{4-[({[4-Chloro-3-(trifluoromethyl)phenyl]amino}carbonyl)amino]phenoxy}-N-methylpyridine-2-carboxamide.The synthesis and use of4-{4-[({[4-Chloro-3-trifluoromethyl)phenyl]amino}carbonyl)amino]phenoxy}-N-methylpyridine-2-carboxamideand many other ureas, as well as pharmaceutically acceptable saltsthereof such as tosylate salts, are described in a number ofapplications including international applications WO 00/42012, WO00/41698, WO 02/062763, WO 03/354,950, WO 02/085859, WO 03/047579 and WO04/15653, which are incorporated herein by reference.

An example of a procedure to synthesizeN-[4-chloro-3-(trifluoromethyl)phenyl]-N′-{4-[2-carbamoyl-1-oxo-(4-pyridyloxy)]phenyl}ureafollows:

Step 1: Preparation of 4-chloro-2-pyridinecarboxamide

To a stirred mixture of methyl 4-chloro-2-pyridinecarboxylatehydrochloride (1.0 g, 4.81 mmol) dissolved in conc. aqueous ammonia (32mL) is added ammonium chloride (96.2 mg, 1.8 mmol, 0.37 equiv.), and theheterogeneous reaction mixture is stirred at ambient temperature for 16h. The reaction mixture is poured into EtOAc (500 mL) and water (300mL). The organic layer is washed with water (2×300 mL) and a saturatedNaCl solution (1×300 mL), dried (MgSO₄), concentrated in vacuo to give4-chloro-2-pyridinecarboxamide as a beige solid (604.3 mg, 80.3%): TLC(50% EtOAc/hexane) R_(f) 0.20; ¹H-NMR (DMSO-d₆) □ 8.61 (d, J=5.4 Hz, 1H), 8.20 (broad s, 1H), 8.02 (d, J=1.8 Hz, 1H), 7.81 (broad s, 1H), 7.76to 7.73 (m, 1H).

Step 2: Preparation of 4-(4-aminophenoxy)-2-pyridinecarboxamide

To 4-aminophenol (418 mg, 3.83 mmol) in anh DMF(7.7 mL) is addedpotassium tert-butoxide (447 mg, 3.98 mmol, 1.04 equiv.) in one portion.The reaction mixture is stirred at room temperature for 2 h, and asolution of 4-chloro-2-pyridinecarboxamide (600 mg, 3.83 mmol, 1.0equiv.) in anh DMF (4 mL) is then added. The reaction mixture is stirredat 80° C. for 3 days and poured into a mixture of EtOAc and a saturatedNaCl solution. The organic layer is sequentially washed with a saturatedNH₄Cl solution then a saturated NaCl solution, dried (MgSO₄), andconcentrated under reduced pressure. The crude product is purified usingMPLC chromatography (Biotage®; gradient from 100% EtOAc to followed by10% MeOH/50% EtOAc/40% hexane) to give the4-chloro-5-trifluoromethylaniline as a brown solid (510 mg, 58%). ¹H-NMR(DMSO-d₆) □ 8.43 (d, J=5.7 Hz, 1 H), 8.07 (br s, I H), 7.66 (br s, 1 H),7.31 (d, J=2.7 Hz, 1 H), 7.07 (dd, J=5.7 Hz, 2.7 Hz, 1 H), 6.85 (d,J=9.0 Hz, 2 H), 6.62 (d, J=8.7 Hz, 2H), 5.17 (broad s, 2H); HPLC EI-MSm/z 230 ((M+H)⁺.

Step 3: Preparation ofN-[4-chloro-3-(trifluoromethyl)phenyl]-N′-{4-[2-carbamoyl-(4-pyridyloxy)]phenyl}urea

A mixture of 4-chloro-5-trifluoromethylaniline (451 mg, 2.31 mmol, 1.1equiv.) and 1,1′-carbonyl diimidazole (419 mg, 2.54 mmol, 1.2 equiv.) inanh dichloroethane (5.5 mL) is stirred under argon at 65° C. for 16 h.Once cooled to room temperature, a solution of4-(4-aminophenoxy)-2-pyridinecarboxamide (480 mg, 2.09 mmol) in anh THF(4.0 mL) is added, and the reaction mixture is stirred at 60° C. for 4h. The reaction mixture is poured into EtOAc, and the organic layer iswashed with water (2×) and a saturated NaCl solution (1×), dried(MgSO₄), filtered, and evaporated in vacuo. Purification using MPLCchromatography (Biotage®; gradient from 100% EtOAc to 2% MeOH/EtOAc)gaveN-[4-chloro-3-(trifluoromethyl)phenyl]-N′-{4-[2-carbamoyl-(4-pyridyloxy)]phenyl}ureaas a white solid (770 mg, 82%): TLC (EtOAc) R_(f) 0.11, 100% ethylacetate ¹H-NMR (DMSO-d₆) □ 9.21 (s, 1H), 8.99 (s, 1H), 8.50 (d, J=5.6Hz, 1H), 8.11 (s, 1H), 8.10 (s, 1H), 7.69 (broad s, 1H), 7.64 (dd, J=8.2Hz, 2.1 Hz, 1H), 7.61 (s, I H), 7.59 (d, J=8.8 Hz, 2H), 7.39 (d, J=2.5Hz, 1H), 7.15 (d, J=8.9 Hz, 2H), 7.14 (m, 1H); MS LC-MS (MH⁺=451). Anal.calcd for C₂₀H₁₄ClF₃N₄O₃: C, 53.29% H, 3.13% N, 12.43%. Found: C, 53.33%H, 3.21% N, 12.60%.

Another method of preparingN-[4-chloro-3-(trifluoromethyl)phenyl]-N′-{4-[2-carbamoyl-(4-pyridyloxy)]phenyl}ureaare described in Bankston et al. “A Scaleable Synthesis of BAY 43-9006:A Potent Raf Kinase Inhibitor for the Treatment of Cancer” Org. Proc.Res. Dev. 2002, 6(6), 777-781.

The formation of pharmaceutically acceptable salts such as tosylatesalts from these ureas can be performed by conventional methods. Anexample of the preparation of sorafenib, the tosylate salt of4-{4-[({[4-Chloro-3-(trifluoromethyl)phenyl]amino}carbonyl)amino]phenoxy}-N-methylpyridine-2-carboxamide,in the polymorph II is as follows:

903 g of4-{4-[({[4-chloro-3-(trifluoromethyl)phenyl]amino}carbonyl)amino]phenoxy-N-methylpyridine-2-carboxamide,prepared as described above, are initially charged in 2700 ml ofethanol. 451.7 g of p-toluenesulfonic acid monohydrate are dissolved in1340 g of ethanol and added dropwise at room temperature. The suspensionis stirred at room temperature for 1 hour, then filtered off withsuction, and the residue is washed three times with 830 ml each time ofethanol. The drying is effected at 50° C. under reduced pressure withsupply of air. 1129.6 g of the tosylate salt of4-{4-[({[4-Chloro-3-(trifluoromethyl)phenyl]amino}carbonyl)amino]phenoxy}-N-methylpyridine-2-carboxamidein the polymorph II are obtained.

An example of the preparation of sorafenib,4-{4-[({[4-chloro-3-(trifluoromethyl)phenyl]amino}carbonyl)-amino]phenoxy}-N-methylpyridine-2-carboxamidetosylate, in the polymorph I is as follows:

-   Heating 5 mg of sorafenib, [tosylate salt of    4-{4-[({[4-chloro-3-(trifluoromethyl)phenyl]amino}carbonyl)amino]-phenoxy}-N-methylpyridine-2-carboxamide]    in the polymorph II to 200° C. at a heating rate of 20° C./min and    subsequently cooling to room temperature at a cooling rate of 2°    C./min. The sample is tested thermoanalytically (DSC) and    corresponds to the title compound in the polymorph I.

Methods for preparing the compounds of this invention are also describedin the following U.S. applications:

-   -   Ser. No. 09/425,228, filed Oct. 22, 1999;    -   Ser. No. 09/722,418 filed Nov. 28, 2000    -   Ser. No. 09/758,547, filed Jan. 12, 2001;    -   Ser. No. 09/838,285, filed Apr. 20, 2001;    -   Ser. No. 09/838,286, filed Apr. 20, 2001; and

The entire disclosure of all applications, patents and publicationscited above and below including copending application Ser. No.10/308,187 filed Dec. 12, 2002 and Ser. No. 10/848,567 filed May 19,2004, are hereby incorporated by reference.

Another embodiment of the present invention is a method for screeningthe effects of a drug on a tissue or cell sample comprising the step ofanalyzing the level of expression of one or more genes and/or geneproducts, wherein the gene expression and/or gene product levels in thetissue or cell sample are analyzed before and after exposure to thedrug, and a variation in the expression level of the gene and/or geneproduct is indicative of a drug effect or provides a patient diagnosisor predicts a patient's response to the treatment. In a furtherembodiment, the drug is sorafenib and/or other diarylureas. In anotherembodiment, the gene or gene product is HER-2, EGFR, VEGF, u-PA,p-PAI-1, and soluble forms thereof, e.g., as detected in tumor tissue,lymph, and/or blood, especially blood serum.

Another aspect of the present invention is a method for discoveringnovel drugs comprising the step of analyzing the level of expression ofone or more genes and/or gene products, wherein the gene expressionand/or gene product levels of the cells are analyzed before and afterexposure to the drug, and a variation in the expression level of thegene and/or gene product is indicative of drug efficacy. In a furtheraspect, the gene or gene product is In another embodiment, the gene orgene product is HER-2, EGFR, VEGF, u-PA, p-PAI-1, and soluble formsthereof, e.g., as detected in tumor tissue, lymph, and/or blood,especially blood serum.

The invention further provides a method for identifying a compounduseful for the treatment of cancer comprising administering to a subjectwith cancer a test compound, and measuring the activity of thepolypeptide, wherein a change in the activity of the polypeptide isindicative of the test compound being useful for the treatment ofcancer. In a further embodiment, the polypeptide is In anotherembodiment, the gene or gene product is HER-2, EGFR, VEGF, u-PA,p-PAI-1, and soluble forms thereof, e.g., as detected in tumor tissue,lymph, and/or blood, especially blood serum, and in another embodiment,the compound is a sorafenib or another diarylurea.

The invention, thus, provides methods which may be used to identifycompounds which may act, for example, as regulators or modulators suchas agonists and antagonists, partial agonists, inverse agonists,activators, co-activators, and inhibitors. Accordingly, the inventionprovides reagents and methods for regulating the expression of apolynucleotide or a polypeptide associated with cancer. Reagents thatmodulate the expression, stability, or amount of a polynucleotide or theactivity of the polypeptide may be a protein, a peptide, apeptidomimetic, a nucleic acid, a nucleic acid analogue (e.g., peptidenucleic acid, locked nucleic acid), or a small molecule.

The present invention also provides a method for providing a patientdiagnosis comprising the step of analyzing the level of expression ofone or more genes and/or gene products, wherein the gene expressionand/or gene product levels of normal and patient samples are analyzed,and a variation in the expression level of the gene and/or gene productin the patient sample is diagnostic of a disease. The patient samplesinclude, but are not limited to, blood, amniotic fluid, plasma, semen,bone marrow, and tissue biopsy. In a further embodiment, the gene orgene product is HER-2, EGFR, VEGF, u-PA, p-PAI-1, and soluble formsthereof, e.g., as detected in tumor tissue, lymph, and/or blood,especially blood serum.

The present invention still further provides a method of diagnosingcancer in a subject comprising measuring the activity of the polypeptidein a subject suspected of having cancer, wherein if there is adifference in the activity of the polypeptide, relative to the activityof the polypeptide in a subject not suspected of having cancer, then thesubject is diagnosed has having cancer. In a further embodiment, thepolypeptide is HER-2, EGFR, VEGF, u-PA, p-PAI-1, and soluble formsthereof, e.g., as detected in tumor tissue, lymph, and/or blood,especially blood serum.

In another embodiment, the invention provides a method for detectingcancer in a patient sample in which an antibody to a protein is used toreact with proteins in the patient sample. In a still furtherembodiment, the antibody is specific for HER-2, EGFR, VEGF, u-PA,p-PAI-1, and soluble forms thereof, e.g., as detected in tumor tissue,lymph, and/or blood, especially blood serum.

Another aspect of the present invention is a method for distinguishingbetween normal and disease states comprising the step of analyzing thelevel of expression of one or more genes and/or gene products, whereinthe gene expression and/or gene product levels of normal and diseasetissues are analyzed, and a variation in the expression level of thegene and/or gene product is indicative of a disease state. In a furtheraspect, the gene or gene product is HER-2, EGFR, VEGF, u-PA, p-PAI-1,and soluble forms thereof, e.g., as detected in tumor tissue, lymph,and/or blood, especially blood serum.

In another embodiment, the invention pertains to a method of determiningthe phenotype of cells comprising detecting the differential expression,relative to normal cells, of at least one gene, wherein the gene isdifferentially expressed by at least a factor of two, at least a factorof five, at least a factor of twenty, or at least a factor of fifty. Ina further embodiment, the gene encodes HER-2, EGFR, VEGF, u-PA, p-PAI-1,and soluble forms thereof.

Any test sample in which it is desired to identify a polynucleotide orpolypeptide thereof can be used, including, e.g., blood, urine, saliva,stool (for extracting nucleic acid, see, e.g., U.S. Pat. No. 6,177,251),swabs comprising tissue, biopsied tissue, tissue sections, culturedcells, etc.

Detection can be accomplished in combination with polynucleotide probesfor other genes, e.g., genes which are expressed in other diseasestates, tissues, cells, such as brain, heart, kidney, spleen, thymus,liver, stomach, small intestine, colon, muscle, lung, testis, placenta,pituitary, thyroid, skin, adrenal gland, pancreas, salivary gland,uterus, ovary, prostate gland, peripheral blood cells (T-cells,lymphocytes, etc.), embryo, normal breast fat, adult and embryonic stemcells, specific cell-types, such as endothelial, epithelial, myocytes,adipose, luminal epithelial, basoepithelial, myoepithelial, stromalcells, etc.

In yet another embodiment, the invention pertains to a method ofdetermining the phenotype of cells, comprising detecting thedifferential expression, relative to normal cells, of at least onepolypeptide, wherein the protein is differentially expressed by at leasta factor of two, at least a factor of five, at least a factor of twenty,an up to at least a factor of fifty. In a further embodiment, thepolypeptide is HER-2, EGFR, VEGF, u-PA, and p-PAI-1.

In another embodiment, the invention pertains to a method fordetermining the phenotype of cells from a patient by providing a nucleicacid probe comprising a nucleotide sequence having at least about 10, atleast about 15, at least about 25, or at least about 40 consecutivenucleotides, obtaining a sample of cells from a patient, optionallyproviding a second sample of cells substantially all of which arenon-cancerous, contacting the nucleic acid probe under stringentconditions with mRNA of each of said first and second cell samples, andcomparing (a) the amount of hybridization of the probe with mRNA of thefirst cell sample, with (b) the amount of hybridization of the probewith mRNA of the second cell sample, wherein a difference of at least afactor of two, at least a factor of five, at least a factor of twenty,or at least a factor of fifty in the amount of hybridization with themRNA of the first cell sample as compared to the amount of hybridizationwith the mRNA of the second cell sample is indicative of the phenotypeof cells in the first cell sample. In a further embodiment, the nucleicacid probe comprises the nucleotide sequence encoding HER-2, EGFR, VEGF,u-PA, p-PAI-1, and soluble forms thereof.

In another embodiment, the invention provides a test kit for identifyingthe presence of cancerous cells or tissues, comprising a probe/primer,for measuring a level of a nucleic acid in a sample of cells or serumisolated from a patient. In certain embodiments, the kit may furtherinclude instructions for using the kit, solutions for suspending orfixing the cells, detectable tags or labels, solutions for rendering anucleic acid susceptible to hybridization, solutions for lysing cells,or solutions for the purification of nucleic acids. In a furtherembodiment, the probe/primer comprises the nucleotide sequence encodingHER-2, EGFR, VEGF, u-PA, p-PAI-1, and soluble forms thereof, e.g., asdetected in tumor tissue, lymph, and/or blood, especially blood serum.

In one embodiment, the invention provides a test kit for identifying thepresence of cancer cells or tissues, comprising an antibody specific fora protein. In certain embodiments, the kit further includes instructionsfor using the kit. In certain embodiments, the kit may further includesolutions for suspending or fixing the cells, detectable tags or labels,solutions for rendering a polypeptide susceptible to the binding of anantibody, solutions for lysing cells, or solutions for the purificationof polypeptides. In a still further embodiment, the antibody is specificfor HER-2, EGFR, VEGF, u-PA, p-PAI-1, and soluble forms thereof

In another embodiment, the invention provides a test kit for monitoringthe efficacy of a compound or therapeutic in cancerous cells or tissues,comprising a probe/primer, for measuring a level of a nucleic acid in asample isolated from a patient. In certain embodiments, the kit mayfurther include instructions for using the kit, solutions for suspendingor fixing the cells, detectable tags or labels, solutions for renderinga nucleic acid susceptible to hybridization, solutions for lysing cells,or solutions for the purification of nucleic acids. In a furtherembodiment, the probe/primer comprises the nucleotide sequence encodingHER-2, EGFR, VEGF, u-PA, p-PAI-1, and soluble forms thereof, e.g., asdetected in tumor tissue, lymph, and/or blood, especially blood serum.

In one embodiment, the invention provides a test kit for monitoring theefficacy of a compound or therapeutic in cancer cells, tissues.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,constructs, and reagents described and as such may vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which will be limited only by theappended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “agene” is a reference to one or more genes and includes equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devices,and materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications and patents mentioned herein are hereby incorporatedherein by reference for the purpose of describing and disclosing, forexample, the constructs and methodologies that are described in thepublications which might be used in connection with the presentlydescribed invention. The publications discussed above and throughout thetext are provided solely for their disclosure prior to the filing dateof the present application. Nothing herein is to be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention.

Definitions

For convenience, the meaning of certain terms and phrases employed inthe specification, examples, and appended claims are provided below.

An “address” on an array (e.g., a microarray) refers to a location atwhich an element, for example, an oligonucleotide, is attached to thesolid surface of the array.

The term “agonist,” as used herein, is meant to refer to an agent thatmimics or up-regulates (e.g., potentiates or supplements) thebioactivity of a protein. An agonist may be a wild-type protein orderivative thereof having at least one bioactivity of the wild-typeprotein. An agonist may also be a compound that up-regulates expressionof a gene or which increases at least one bioactivity of a protein. Anagonist can also be a compound which increases the interaction of apolypeptide with another molecule, for example, a target peptide ornucleic acid.

“Amplification,” as used herein, relates to the production of additionalcopies of a nucleic acid sequence. For example, amplification may becarried out using polymerase chain reaction (PCR) technologies which arewell known in the art. (see, e.g., Dieffenbach and Dveksler (1995) PCRPrimer, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.)

The term “antibody,” as used herein, is intended to include wholeantibodies, for example, of any isotype (IgG, IgA, IgM, IgE, etc.), andincludes fragments thereof which are also specifically reactive with avertebrate (e.g., mammalian) protein. Antibodies may be fragmented usingconventional techniques and the fragments screened for utility in thesame manner as described above for whole antibodies. Thus, the termincludes segments of proteolytically-cleaved or recombinantly-preparedportions of an antibody molecule that are capable of selectivelyreacting with a certain protein. Non-limiting examples of suchproteolytic and/or recombinant fragments include Fab, F(ab′)2, Fab′, Fv,and single chain antibodies (scFv) containing a V[L] and/or V[H] domainjoined by a peptide linker. The scFv's may be covalently ornon-covalently linked to form antibodies having two or more bindingsites. The subject invention includes polyclonal, monoclonal, human,single chain, humanized, and other antibody types and recombinantantibodies.

The terms “array” or “matrix” refer to an arrangement of addressablelocations or “addresses” on a device. The locations can be arranged intwo-dimensional arrays, three-dimensional arrays, or other matrixformats. The number of locations may range from several to at leasthundreds of thousands. Most importantly, each location represents atotally independent reaction site. A “nucleic acid array” refers to anarray containing nucleic acid probes, such as oligonucleotides or largerportions of genes. The nucleic acid on the array is preferablysingle-stranded. Arrays wherein the probes are oligonucleotides arereferred to as “oligonucleotide arrays” or “oligonucleotide chips.” A“microarray,” also referred to herein as a “biochip” or “biologicalchip,” is an array of regions having a density of discrete regions of atleast about 100/cm², and preferably at least about 1000/cm². The regionsin a microarray have typical dimensions, for example, diameters, in therange of between about 10-250 μm, and are separated from other regionsin the array by about the same distance.

“Biological activity” or “bioactivity” or “activity” or “biologicalfunction,” which are used interchangeably, herein mean an effector orantigenic function that is directly or indirectly performed by apolypeptide (whether in its native or denatured conformation), or by anysubsequence thereof. Biological activities include binding topolypeptides, binding to other proteins or molecules, activity as a DNAbinding protein, as a transcription regulator, ability to bind damagedDNA, etc. A bioactivity can be modulated by directly affecting thesubject polypeptide. Alternatively, a bioactivity can be altered bymodulating the level of the polypeptide, such as by modulatingexpression of the corresponding gene.

The term “biological sample,” as used herein, refers to a sampleobtained from an organism or from components (e.g., cells) of anorganism. The sample may be of any biological tissue or fluid. Thesample may be a sample which is derived from a patient. Such samplesinclude, but are not limited to, sputum, blood, blood cells (e.g., whiteblood cells), tissue or biopsy samples (e.g., tumor biopsy), urine,peritoneal fluid, and pleural fluid, or cells therefrom. Biologicalsamples may also include sections of tissues such as frozen sectionstaken for histological purposes.

The term “biomarker” or “marker” encompasses a broad range of intra- andextra-cellular events as well as whole-organism physiological changes.Biomarkers may be represent essentially any aspect of cell function, forexample, but not limited to, levels or rate of production of signalingmolecules, transcription factors, metabolites, gene transcripts as wellas post-translational modifications of proteins. Biomarkers may includewhole genome analysis of transcript levels or whole proteome analysis ofprotein levels and/or modifications.

A biomarker may also refer to a gene or gene product which is up- ordown-regulated in a compound-treated, diseased cell of a subject havingthe disease compared to an untreated diseased cell. That is, the gene orgene product is sufficiently specific to the treated cell that it may beused, optionally with other genes or gene products, to identify,predict, or detect efficacy of a small molecule. Thus, a biomarker is agene or gene product that is characteristic of efficacy of a compound ina diseased cell or the response of that diseased cell to treatment bythe compound.

A nucleotide sequence is “complementary” to another nucleotide sequenceif each of the bases of the two sequences match, that is, are capable offorming Watson-Crick base pairs. The term “complementary strand” is usedherein interchangeably with the term “complement.” The complement of anucleic acid strand may be the complement of a coding strand or thecomplement of a non-coding strand.

“Detection agents of genes” refers to agents that can be used tospecifically detect the gene or other biological molecules relating toit, for example, RNA transcribed from the gene or polypeptides encodedby the gene. Exemplary detection agents are nucleic acid probes, whichhybridize to nucleic acids corresponding to the gene, and antibodies.

Baseline levels can refer to a standard control for “normal” levels(i.e., patients without disease), but can also be comparative, e.g.,where low baseline levels is compared to the levels of other subjectshaving the disease.

The term “cancer” includes, but is not limited to, solid tumors, such ascancers of the breast, respiratory tract, brain, reproductive organs,digestive tract, urinary tract, eye, liver, skin, head and neck,thyroid, parathyroid, and their distant metastases. The term alsoincludes lymphomas, sarcomas, and leukemias.

Examples of breast cancer include, but are not limited to, invasiveductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ,and lobular carcinoma in situ.

Examples of cancers of the respiratory tract include, but are notlimited to, small-cell and non-small-cell lung carcinoma, as well asbronchial adenoma and pleuropulmonary blastoma.

Examples of brain cancers include, but are not limited to, brain stemand hypophtalmic glioma, cerebellar and cerebral astrocytoma,medulloblastoma, ependymoma, as well as neuroectodermal and pinealtumor.

Tumors of the male reproductive organs include, but are not limited to,prostate and testicular cancer. Tumors of the female reproductive organsinclude, but are not limited to, endometrial, cervical, ovarian,vaginal, and vulvar cancer, as well as sarcoma of the uterus.

Tumors of the digestive tract include, but are not limited to, anal,colon, colorectal, esophageal, gallbladder, gastric, pancreatic, rectal,small-intestine, and salivary gland cancers.

Tumors of the urinary tract include, but are not limited to, bladder,penile, kidney, renal pelvis, ureter, and urethral cancers.

Eye cancers include, but are not limited to, intraocular melanoma andretinoblastoma.

Examples of liver cancers include, but are not limited to,hepatocellular carcinoma (liver cell carcinomas with or withoutfibrolamellar variant), cholangiocarcinoma (intrahepatic bile ductcarcinoma), and mixed hepatocellular cholangiocarcinoma.

Skin cancers include, but are not limited to, squamous cell carcinoma,Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, andnon-melanoma skin cancer.

Head-and-neck cancers include, but are not limited to,laryngeal/hypopharyngeal/ nasopharyngeal/oropharyngeal cancer, and lipand oral cavity cancer.

Lymphomas include, but are not limited to, AIDS-related lymphoma,non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease,and lymphoma of the central nervous system.

Sarcomas include, but are not limited to, sarcoma of the soft tissue,osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, andrhabdomyosarcoma.

Leukemias include, but are not limited to, acute myeloid leukemia, acutelymphoblastic leukemia, chronic lymphocytic leukemia, chronicmyelogenous leukemia, and hairy cell leukemia.

“A diseased cell of cancer” refers to a cell present in subjects havingcancer. That is, a cell which is a modified form of a normal cell and isnot present in a subject not having cancer, or a cell which is presentin significantly higher or lower numbers in subjects having cancerrelative to subjects not having cancer.

The term “equivalent” is understood to include nucleotide sequencesencoding functionally equivalent polypeptides. Equivalent nucleotidesequences may include sequences that differ by one or more nucleotidesubstitutions, additions, or deletions, such as allelic variants.

The term “expression profile,” which is used interchangeably herein with“gene expression profile” and “fingerprint” of a cell refers to a set ofvalues representing mRNA levels of one or more genes in a cell. Anexpression profile preferably comprises values representing expressionlevels of at least about 10 genes, preferably at least about 50, 100,200 or more genes. Expression profiles may also comprise an mRNA levelof a gene which is expressed at similar levels in multiple cells andconditions (e.g., a housekeeping gene such as GAPDH). For example, anexpression profile of a diseased cell of cancer refers to a set ofvalues representing mRNA levels of 10 or more genes in a diseased cell.

The term “gene” refers to a nucleic acid sequence that comprises controland coding sequences necessary for the production of a polypeptide orprecursor. The polypeptide can be encoded by a full length codingsequence or by any portion of the coding sequence. The gene may bederived in whole or in part from any source known to the art, includinga plant, a fungus, an animal, a bacterial genome or episome, eukaryotic,nuclear or plasmid DNA, cDNA, viral DNA, or chemically synthesized DNA.A gene may contain one or more modifications in either the coding or theuntranslated regions which could affect the biological activity or thechemical structure of the expression product, the rate of expression, orthe manner of expression control. Such modifications include, but arenot limited to, mutations, insertions, deletions, and substitutions ofone or more nucleotides. The gene may constitute an uninterrupted codingsequence or it may include one or more introns, bound by the appropriatesplice junctions.

“Hybridization” refers to any process by which a strand of nucleic acidbinds with a complementary strand through base pairing. For example, twosingle-stranded nucleic acids “hybridize” when they form adouble-stranded duplex. The region of double-strandedness may includethe full-length of one or both of the single-stranded nucleic acids, orall of one single-stranded nucleic acid and a subsequence of the othersingle-stranded nucleic acid, or the region of double-strandedness mayinclude a subsequence of each nucleic acid. Hybridization also includesthe formation of duplexes which contain certain mismatches, providedthat the two strands are still forming a double-stranded helix.“Stringent hybridization conditions” refers to hybridization conditionsresulting in essentially specific hybridization.

The term “isolated,” as used herein, with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs or RNAs,respectively, that are present in the natural source of themacromolecule. The term “isolated” as used herein also refers to anucleic acid or peptide that is substantially free of cellular material,viral material, culture medium when produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemicallysynthesized. Moreover, an “isolated nucleic acid” may include nucleicacid fragments which are not naturally occurring as fragments and wouldnot be found in the natural state. The term “isolated” is also usedherein to refer to polypeptides which are isolated from other cellularproteins and is meant to encompass both purified and recombinantpolypeptides.

As used herein, the terms “label” and “detectable label” refer to amolecule capable of detection, including, but not limited to,radioactive isotopes, fluorophores, chemiluminescent moieties, enzymes,enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metalions, ligands (e.g., biotin or haptens), and the like. The term“fluorescer” refers to a substance or a portion thereof which is capableof exhibiting fluorescence in the detectable range. Particular examplesof labels which may be used in the present invention includefluorescein, rhodamine, dansyl, umbelliferone, Texas red, luminol,NADPH, alpha-beta-galactosidase, and horseradish peroxidase.

As used herein, the term “level of expression” refers to the measurableexpression level of a given nucleic acid. The level of expression of anucleic acid is determined by methods well known in the art. The term“differentially expressed” or “differential expression” refers to anincrease or decrease in the measurable expression level of a givennucleic acid. As used herein, “differentially expressed” or“differential expression” means the difference in the level ofexpression of a nucleic acid is at least 1.4-fold or more in two samplesused for comparison, both of which are compared to the same normalstandard sample. “Differentially expressed” or “differential expression”according to the invention also means a 1.4-fold, or more, up to andincluding 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or more differencein the level of expression of a nucleic acid in two samples used forcomparison. A nucleic acid is also said to be “differentially expressed”in two samples if one of the two samples contains no detectableexpression of a given nucleic acid, provided that the detectablyexpressed nucleic acid is expressed at +/− at least 1.4 fold.Differential expression of a nucleic acid sequence is “inhibited” thedifference in the level of expression of the nucleic acid in two or moresamples used for comparison is altered such that it is no longer atleast a 1.4 fold difference. Absolute quantification of the level ofexpression of a nucleic acid may be accomplished by including a knownconcentration(s) of one or more control nucleic acid species, generatinga standard curve based on the amount of the control nucleic acid andextrapolating the expression level of the “unknown” nucleic acid speciesfrom the hybridization intensities of the unknown with respect to thestandard curve.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA) and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs and, asapplicable to the embodiment being described, single-stranded (sense orantisense) and double-stranded polynucleotides. Chromosomes, cDNAs,mRNAs, rRNAs, and ESTs are representative examples of molecules that maybe referred to as nucleic acids.

The term “oligonucleotide” as used herein refers to a nucleic acidmolecule comprising, for example, from about 10 to about 1000nucleotides. Oligonucleotides for use in the present invention arepreferably from about 15 to about 150 nucleotides, more preferably fromabout 150 to about 1000 in length. The oligonucleotide may be anaturally occurring oligonucleotide or a synthetic oligonucleotide.Oligonucleotides may be prepared by the phosphoramidite method (Beaucageand Carruthers, Tetrahedron Lett. 22:1859-62, 1981), or by the triestermethod (Matteucci, et al., J. Am. Chem. Soc. 103:3185, 1981), or byother chemical methods known in the art.

The term “patient” or “subject” as used herein includes mammals (e.g.,humans and animals).

As used herein, a nucleic acid or other molecule attached to an array isreferred to as a “probe” or “capture probe.” When an array containsseveral probes corresponding to one gene, these probes are referred toas a “gene-probe set.” A gene-probe set may consist of, for example,about 2 to about 20 probes, preferably from about 2 to about 10 probes,and most preferably about 5 probes.

The “profile” of a cell's biological state refers to the levels ofvarious constituents of a cell that are known to change in response todrug treatments and other perturbations of the biological state of thecell. Constituents of a cell include, for example, levels of RNA, levelsof protein abundances, or protein activity levels.

The term “protein” is used interchangeably herein with the terms“peptide” and “polypeptide.”

An expression profile in one cell is “similar” to an expression profilein another cell when the level of expression of the genes in the twoprofiles are sufficiently similar that the similarity is indicative of acommon characteristic, for example, the same type of cell. Accordingly,the expression profiles of a first cell and a second cell are similarwhen at least 75% of the genes that are expressed in the first cell areexpressed in the second cell at a level that is within a factor of tworelative to the first cell.

“Small molecule,” as used herein, refers to a composition with amolecular weight of less than about 5 kD and most preferably less thanabout 4 kD. Small molecules can be nucleic acids, peptides,polypeptides, peptidomimetics, carbohydrates, lipids, or other organicor inorganic molecules. Many pharmaceutical companies have extensivelibraries of chemical and/or biological mixtures, often fungal,bacterial, or algal extracts, which can be screened with any of theassays of the invention to identify compounds that modulate abioactivity.

The term “specific hybridization” of a probe to a target site of atemplate nucleic acid refers to hybridization of the probe predominantlyto the target, such that the hybridization signal can be clearlyinterpreted. As further described herein, such conditions resulting inspecific hybridization vary depending on the length of the region ofhomology, the GC content of the region, and the melting temperature(“Tm”) of the hybrid. Thus, hybridization conditions may vary in saltcontent, acidity, and temperature of the hybridization solution and thewashes.

A “variant” of polypeptide refers to a polypeptide having an amino acidsequence in which one or more amino acid residues is altered. Thevariant may have “conservative” changes, wherein a substituted aminoacid has similar structural or chemical properties (e.g., replacement ofleucine with isoleucine). A variant may also have “nonconservative”changes (e.g., replacement of glycine with tryptophan). Analogous minorvariations may include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be identified using computer programs well known in theart, for example, LASERGENE software (DNASTAR).

The term “variant,” when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to that of aparticular gene or the coding sequence thereof. This definition may alsoinclude, for example, “allelic,” “splice,” “species,” or “polymorphic”variants. A splice variant may have significant identity to a referencemolecule, but will generally have a greater or lesser number ofpolynucleotides due to alternate splicing of exons during mRNAprocessing. The corresponding polypeptide may possess additionalfunctional domains or an absence of domains. Species variants arepolynucleotide sequences that vary from one species to another. Theresulting polypeptides generally will have significant amino acididentity relative to each other. A polymorphic variant is a variation inthe polynucleotide sequence of a particular gene between individuals ofa given species. Polymorphic variants also may encompass “singlenucleotide polymorphisms” (SNPs) in which the polynucleotide sequencevaries by one base. The presence of SNPs may be indicative of, forexample, a certain population, a disease state, or a propensity for adisease state.

An aspect of the invention is directed to the identification of agentscapable of modulating the differentiation and proliferation of cellscharacterized by aberrant proliferation. More specifically, theinvention relates to methods of screening candidate compounds orsubstances for their ability to regulate the differential expression ofnucleic acid sequences. That is, if a nucleic acid sequence isoverexpressed in cancer cells, then the candidate compounds are screenedfor their ability to decrease expression, and if a nucleic acid sequenceis under-expressed in cancer cells, then a test compound is screened forits ability to increase expression. In addition, the invention relatesto screening assays to identify test compounds or substances whichmodulate the activity of one or more polypeptides which are encoded bythe differentially expressed sequences described herein. In this regard,the invention provides assays for determining compounds that modulatethe expression of marker nucleic acids and/or alter the bioactivity ofthe encoded polypeptide.

Screening for Modulation of Differential Expression

Drug screening is performed by adding a test compound (e.g., sorafenib)to a sample of cells, and monitoring the effect. A parallel sample whichdoes not receive the test compound is also monitored as a control. Thetreated and untreated cells are then compared by any suitable phenotypiccriteria, including but not limited to microscopic analysis, viabilitytesting, ability to replicate, histological examination, the level of aparticular RNA or polypeptide associated with the cells, the level ofenzymatic activity expressed by the cells or cell lysates, and theability of the cells to interact with other cells or compounds.Differences between treated and untreated cells indicates effectsattributable to the test compound.

Desirable effects of a test compound include an effect on any phenotypethat was conferred by the cancer-associated marker nucleic acidsequence. Examples include a test compound that limits the overabundanceof mRNA, limits production of the encoded protein, or limits thefunctional effect of the protein. The effect of the test compound wouldbe apparent when comparing results between treated and untreated cells.

The invention thus, also encompasses methods of screening for agents(e.g., sorafenib which inhibit or enhance the expression of the nucleicacid markers in vitro, comprising exposing a cell or tissue in which themarker nucleic acid mRNA or protein (e.g., HER-2 or VEGF) is detectablein cultured cells to an agent in order to determine whether the agent iscapable of inhibiting or enhancing production of the mRNA; anddetermining the level of mRNA in the exposed cells or tissue, wherein adecrease in the level of the mRNA after exposure of the cell line to theagent is indicative of inhibition of the marker nucleic acid mRNAproduction and an increase in mRNA levels is indicative of enhancementof maker mRNA production.

Alternatively, the screening method may include in vitro screening of acell or tissue in which marker protein is detectable in cultured cellsto an agent suspected of inhibiting or enhancing production of themarker protein; and determining the level of the marker protein in thecells or tissue, wherein a decrease in the level of marker protein afterexposure of the cells or tissue to the agent is indicative of inhibitionof marker protein production and an increase on the level of markerprotein is indicative of enhancement of marker protein production.

The invention also encompasses in vivo methods of screening for agentswhich inhibit or enhance expression of the marker nucleic acids,comprising exposing a subject having tumor cells in which marker mRNA orprotein is detectable to an agent suspected of inhibiting or enhancingproduction of marker mRNA or protein; and determining the level ofmarker mRNA or protein in tumor cells of the exposed mammal. A decreasein the level of marker mRNA or protein after exposure of the subject tothe agent is indicative of inhibition of marker nucleic acid expressionand an increase in the level of marker mRNA or protein is indicative ofenhancement of marker nucleic acid expression.

Accordingly, the invention provides a method comprising incubating acell expressing the marker nucleic acids with a test compound andmeasuring the mRNA or protein level. The invention further provides amethod for quantitatively determining the level of expression of themarker nucleic acids in a cell population, and a method for determiningwhether an agent is capable of increasing or decreasing the level ofexpression of the marker nucleic acids in a cell population. The methodfor determining whether an agent is capable of increasing or decreasingthe level of expression of the marker nucleic acids in a cell populationcomprises the steps of (a) preparing cell extracts from control andagent-treated cell populations, (b) isolating the marker polypeptidesfrom the cell extracts, and (c) quantifying (e.g., in parallel) theamount of an immunocomplex formed between the marker polypeptide and anantibody specific to said polypeptide. The marker polypeptides of thisinvention may also be quantified by assaying for its bioactivity. Agentsthat induce an increase in the marker nucleic acid expression may beidentified by their ability to increase the amount of immunocomplexformed in the treated cell as compared with the amount of theimmunocomplex formed in the control cell. In a similar manlier, agentsthat decrease expression of the marker nucleic acid may be identified bytheir ability to decrease the amount of the immunocomplex formed in thetreated cell extract as compared to the control cell.

The present invention provides isolated nucleic acid sequences which aredifferentially regulated in cancer, and a method for identifying suchsequences. The present invention provides a method for identifying anucleotide sequence which is differentially regulated in a subject withcancer, comprising: hybridizing a nucleic acid sample corresponding toRNA obtained from the subject to a nucleic acid sample comprising one ormore nucleic acid molecules of known identity; and measuring thehybridization of the nucleic acid sample to the one or more nucleic acidmolecules of known identity, wherein a two-fold difference in thehybridization of the nucleic acid sample to the one or more nucleic acidmolecules of known identity relative to a nucleic acid sample obtainedfrom a subject without cancer is indicative of the differentialexpression of the nucleotide sequence in a subject with cancer.

Generally, the present invention provides a method for identifyingnucleic acid sequences which are differentially regulated in a subjectwith cancer comprising isolating messenger RNA from a subject,generating cRNA from the mRNA sample, hybridizing the cRNA to amicroarray comprising a plurality of nucleic acid molecules stablyassociated with discrete locations on the array, and identifyingpatterns of hybridization of the cRNA to the array. According to thepresent invention, a nucleic acid molecule which hybridizes to a givenlocation on the array is said to be differentially regulated if thehybridization signal is at least two-fold higher or lower than thehybridization signal at the same location on an identical arrayhybridized with a nucleic acid sample obtained from a subject that doesnot have cancer.

Microarrays for Determining the Level of Expression of Genes

Determining gene expression levels may be accomplished utilizingmicroarrays. Generally, the following steps may be involved: (a)obtaining an mRNA sample from a subject and preparing labeled nucleicacids therefrom (the “target nucleic acids” or “targets”); (b)contacting the target nucleic acids with an array under conditionssufficient for the target nucleic acids to bind to the correspondingprobes on the array, for example, by hybridization or specific binding;(c) optional removal of unbound targets from the array; (d) detectingthe bound targets, and (e) analyzing the results, for example, usingcomputer based analysis methods. As used herein, “nucleic acid probes”or “probes” are nucleic acids attached to the array, whereas “targetnucleic acids” are nucleic acids that are hybridized to the array.

Nucleic acid specimens may be obtained from a subject to be tested usingeither “invasive” or “non-invasive” sampling means. A sampling means issaid to be “invasive” if it involves the collection of nucleic acidsfrom within the skin or organs of an animal (including murine, human,ovine, equine, bovine, porcine, canine, or feline animal). Examples ofinvasive methods include, for example, blood collection, semencollection, needle biopsy, pleural aspiration, umbilical cord biopsy.Examples of such methods are discussed by Kim, et al., (J. Virol.66:3879-3882, 1992); Biswas, et al., (Ann. NY Acad. Sci. 590:582-583,1990); and Biswas, et al., (J. Clin. Microbial. 29:2228-2233, 1991).

In contrast, a “non-invasive” sampling means is one in which the nucleicacid molecules are recovered from an internal or external surface of theanimal. Examples of such “non-invasive” sampling means include, forexample, “swabbing,” collection of tears, saliva, urine, fecal material,sweat or perspiration, hair.

In one embodiment of the present invention, one or more cells from thesubject to be tested are obtained and RNA is isolated from the cells. Ina preferred embodiment, a sample of peripheral blood leukocytes (PBLs)cells is obtained from the subject. It is also possible to obtain a cellsample from a subject, and then to enrich the sample for a desired celltype. For example, cells may be isolated from other cells using avariety of techniques, such as isolation with an antibody binding to anepitope on the cell surface of the desired cell type. Where the desiredcells are in a solid tissue, particular cells may be dissected, forexample, by microdissection or by laser capture microdissection (LCM)(see, e.g., Bonner, et al., Science 278:1481, 1997; Emmert-Buck, et al.,Science 274:998, 1996; Fend, et al., Am. J. Path. 154:61, 1999; andMurakami, et al., Kidney Int. 58:1346, 2000).

RNA may be extracted from tissue or cell samples by a variety ofmethods, for example, guanidium thiocyanate lysis followed by CsClcentrifugation (Chirgwin, et al., Biochemistry 18:5294-5299, 1979). RNAfrom single cells may be obtained as described in methods for preparingcDNA libraries from single cells (see, e.g., Dulac, Curr. Top. Dev.Biol. 36:245, 1998; Jena, et al., J. Immunol. Methods 190:199, 1996).

The RNA sample can be further enriched for a particular species. In oneembodiment, for example, poly(A)+ RNA may be isolated from an RNAsample. In another embodiment, the RNA population may be enriched forsequences of interest by primer-specific cDNA synthesis, or multiplerounds of linear amplification based on cDNA synthesis andtemplate-directed in vitro transcription (see, e.g., Wang, et al., Proc.Natl. Acad. Sci. USA 86:9717, 1989; Dulac, et al., supra; Jena, et al.,supra). In addition, the population of RNA, enriched or not inparticular species or sequences, may be further amplified by a varietyof amplification methods including, for example, PCR; ligase chainreaction (LCR) (see, e.g., Wu and Wallace, Genomics 4:560, 1989;Landegren, et al., Science 241:1077, 1988); self-sustained sequencereplication (SSR) (see, e.g., Guatelli, et al., Proc. Natl. Acad. Sci.USA 87:1874, 1990); nucleic acid based sequence amplification (NASBA)and transcription amplification (see, e.g., Kwoh, et al., Proc. Natl.Acad. Sci. USA 86:1173, 1989). Methods for PCR technology are well knownin the art (see, e.g., PCR Technology: Principles and Applications forDNA Amplification (ed. H. A. Erlich, Freeman Press, N.Y., N.Y., 1992);PCR Protocols: A Guide to Methods and Applications (eds. Innis, et al.,Academic Press, San Diego, Calif., 1990); Mattila, et al., Nucleic AcidsRes. 19:4967, 1991; Eckert, et al., PCR Methods and Applications 1:17,1991; PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. No.4,683,202). Methods of amplification are described, for example, byOhyama, et al., (BioTechniques 29:530, 2000); Luo, et al., (Nat. Med.5:117, 1999); Hegde, et al., (BioTechniques 29:548, 2000); Kacharmina,et al., (Meth. Enzymol. 303:3, 1999); Livesey, et al., Curr. Biol.10:301, 2000); Spirin, et al., (Invest. Ophtalmol. Vis. Sci. 40:3108,1999); and Sakai, et at., (Anal. Biochem. 287:32, 2000). RNAamplification and cDNA synthesis may also be conducted in cells in situ(see, e.g., Eberwine, et al. Proc. Natl. Acad. Sci. USA 89:3010, 1992).

The nucleic acid molecules may be labeled to permit detection ofhybridization of the nucleic acid molecules to a microarray. That is,the probe may comprise a member of a signal producing system and thus,is detectable, either directly or through combined action with one ormore additional members of a signal producing system. For example, thenucleic acids may be labeled with a fluorescently labeled dNTP (see,e.g., Kricka, 1992, Nonisotopic DNA Probe Techniques, Academic Press SanDiego, Calif.), biotinylated dNTPs or rNTP followed by addition oflabeled streptavidin, chemiluminescent labels, or isotopes. Anotherexample of labels include “molecular beacons” as described in Tyagi andKramer (Nature Biotech. 14:303, 1996). Hybridization may be also bedetermined, for example, by plasmon resonance (see, e.g., Thiel, et al.Anal. Chem. 69:4948, 1997).

In one embodiment, a plurality (e.g., 2, 3, 4, 5, or more) of sets oftarget nucleic acids are labeled and used in one hybridization reaction(“multiplex” analysis). For example, one set of nucleic acids maycorrespond to RNA from one cell and another set of nucleic acids maycorrespond to RNA from another cell. The plurality of sets of nucleicacids may be labeled with different labels, for example, differentfluorescent labels (e.g., fluorescein and rhodamine) which have distinctemission spectra so that they can be distinguished. The sets may then bemixed and hybridized simultaneously to one microarray (see, e.g., Shena,et al., Science 270:467-470, 1995).

Microarrays for use according to the invention include one or moreprobes of genes characteristic of small molecule efficacy. In apreferred embodiment, the microarray comprises probes corresponding toone or more of genes selected from the group consisting of genes whichare up-regulated in cancer and genes which are down-regulated in cancer.The microarray may comprise probes corresponding to at least 10,preferably at least 20, at least 50, at least 100 or at least 1000 genescharacteristic of small molecule efficacy.

There may be one or more than one probe corresponding to each gene on amicroarray. For example, a microarray may contain from 2 to 20 probescorresponding to one gene and preferably about 5 to 10. The probes maycorrespond to the full-length RNA sequence or complement thereof ofgenes characteristic of small molecule efficacy, or the probe maycorrespond to a portion thereof, which portion is of sufficient lengthto permit specific hybridization. Such probes may comprise from about 50nucleotides to about 100, 200, 500, or 1000 nucleotides or more than1000 nucleotides. As further described herein, microarrays may containoligonucleotide probes, consisting of about 10 to 50 nucleotides,preferably about 15 to 30 nucleotides and more preferably about 20-25nucleotides. The probes are preferably single-stranded and will havesufficient complementarity to its target to provide for the desiredlevel of sequence specific hybridization.

Typically, the arrays used in the present invention will have a sitedensity of greater than 100 different probes per cm². Preferably, thearrays will have a site density of greater than 500/cm², more preferablygreater than about 1000/cm², and most preferably, greater than about10,000/cm². Preferably, the arrays will have more than 100 differentprobes on a single substrate, more preferably greater than about 1000different probes, still more preferably, greater than about 10,000different probes and most preferably, greater than 100,000 differentprobes on a single substrate.

A number of different microarray configurations and methods for theirproduction are known to those of skill in the art and are disclosed inU.S. Pat. Nos: 5,242,974; 5,384,261; 5,405,783; 5,412,087; 5,424,186;5,429,807; 5,436,327; 5,445,934; 5,556,752; 5,405,783; 5,412,087;5,424,186; 5,429,807; 5,436,327; 5,472,672; 5,527,681; 5,529,756;5,545,531; 5,554,501; 5,561,071; 5,571,639; 5,593,839; 5,624,711;5,700,637; 5,744,305; 5,770,456; 5,770,722; 5,837,832; 5,856,101;5,874,219; 5,885,837; 5,919,523; 6,022,963; 6,077,674; and 6,156,501;Shena, et al., Tibtech 16:301, 1998; Duggan, et al., Nat. Genet. 21:10,1999; Bowtell, et al., Nat. Genet. 21:25, 1999; Lipshutz, et al., 21Nature Genet. 20-24, 1999; Blanchard, et al., 11 Biosensors andBioelectronics, 687-90, 1996; Maskos, et al., 21 Nucleic Acids Res.4663-69, 1993; Hughes, et al., Nat. Biotechol. (2001) 19:342; thedisclosures of which are herein incorporated by reference. Patentsdescribing methods of using arrays in various applications include: U.S.Pat. Nos. 5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710;5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732;5,661,028; 5,848,659; and 5,874,219; the disclosures of which are hereinincorporated by reference.

Arrays preferably include control and reference nucleic acids. Controlnucleic acids include, for example, prokaryotic genes such as bioB, bioCand bioD, cre from P1 bacteriophage or polyA controls, such as dap, lys,phe, thr, and trp. Reference nucleic acids allow the normalization ofresults from one experiment to another and the comparison of multipleexperiments on a quantitative revel. Exemplary reference nucleic acidsinclude housekeeping genes of known expression levels, for example,GAPDH, hexokinase, and actin.

In one embodiment, an array of oligonucleotides may be synthesized on asolid support. Exemplary solid supports include glass, plastics,polymers, metals, metalloids, ceramics, organics, etc. Using chipmasking technologies and photoprotective chemistry, it is possible togenerate ordered arrays of nucleic acid probes. These arrays, which areknown, for example, as “DNA chips” or very large scale immobilizedpolymer arrays (“VLSIPS™” arrays), may include millions of defined proberegions on a substrate having an area of about 1 cm² to several cm²,thereby incorporating from a few to millions of probes (see, e.g., U.S.Pat. No. 5,631,734).

To compare expression levels, labeled nucleic acids may be contactedwith the array under conditions sufficient for binding between thetarget nucleic acid and the probe on the array. In a preferredembodiment, the hybridization conditions may be selected to provide forthe desired level of hybridization specificity; that is, conditionssufficient for hybridization to occur between the labeled nucleic acidsand probes on the microarray.

Hybridization may be carried out in conditions permitting essentiallyspecific hybridization. The length and GC content of the nucleic acidwill determine the thermal melting point and thus, the hybridizationconditions necessary for obtaining specific hybridization of the probeto the target nucleic acid. These factors are well known to a person ofskill in the art, and may also be tested in assays. An extensive guideto nucleic acid hybridization may be found in Tijssen, et al.(Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24:Hybridization With Nucleic Acid Probes, P. Tijssen, ed. Elsevier, N.Y.,(1993)).

The methods described above result in the production of hybridizationpatterns of labeled target nucleic acids on the array surface. Theresultant hybridization patterns of labeled nucleic acids may bevisualized or detected in a variety of ways, with the particular mannerof detection selected based on the particular label of the targetnucleic acid. Representative detection means include scintillationcounting, autoradiography, fluorescence measurement, colorimetricmeasurement, light emission measurement, light scattering, and the like.

One such method of detection utilizes an array scanner that iscommercially available (Affymetrix, Santa Clara, Calif.), for example,the 417™ Arrayer, the 418™ Array Scanner, or the Agilent GeneArray™Scanner. This scanner is controlled from a system computer with aninterface and easy-to-use software tools. The output may be directlyimported into or directly read by a variety of software applications.Preferred scanning devices are described in, for example, U.S. Pat. Nos.5,143,854 and 5,424,186.

For fluorescent labeled probes, the fluorescence emissions at each siteof a transcript array may be, preferably, detected by scanning confocallaser microscopy. Alternatively, a laser may be used that allowssimultaneous specimen illumination at wavelengths specific to the twofluorophores and emissions from the two fluorophores may be analyzedsimultaneously (see, e.g., Shalon, et al., Genome Res. 6:639-645, 1996).In a preferred embodiment, the arrays may be scanned with a laserfluorescent scanner with a computer controlled X-Y stage and amicroscope objective. Fluorescence laser scanning devices are describedin Shalon, et al., supra.

Various algorithms are available for analyzing gene expression data, forexample, the type of comparisons to perform. In certain embodiments, itis desirable to group genes that are co-regulated. This allows for thecomparison of large numbers of profiles. A preferred embodiment foridentifying such groups of genes involves clustering algorithms (forreviews of clustering algorithms, see, e.g., Fukunaga, 1990, StatisticalPattern Recognition, 2nd Ed., Academic Press, San Diego; Everitt, 1974,Cluster Analysis, London: Heinemann Educ. Books; Hartigan, 1975,Clustering Algorithms, New York: Wiley; Sneath and Sokal, 1973,Numerical Taxonomy, Freeman; Anderberg, 1973, Cluster Analysis forApplications, Academic Press: New York).

Biomarker Discovery

Expression patterns may be used to derive a panel of biomarkers that canbe used to predict the efficacy of drug treatment in the patients. Thebiomarkers may consist of gene expression levels from microarrayexperiments on RNA isolated from biological samples, RNA isolated fromfrozen samples of tumor biopsies, or mass spectrometry-derived proteinmasses in the serum.

Although the precise mechanism for data analysis will depend upon theexact nature of the data, a typical procedure for developing a panel ofbiomarkers is as follows. The data (gene expression levels or massspectra) are collected for each patient prior to treatment. As the studyprogresses, the patients are classified according to their response tothe drug treatment; either as efficacious or non-efficacious. Multiplelevels of efficacy can be accommodated in a data model, but a binarycomparison is considered optimal, particularly if the patient populationis less than several hundred. Assuming adequate numbers of patients ineach class, the protein and/or gene expression data may be analyzed by anumber of techniques known in the art. Many of the techniques arederived from traditional statistics as well from the field of machinelearning. These techniques serve two purposes:

1. Reduce the dimensionality of data—In the case of mass spectra or geneexpression microarrays, data is reduced from many thousands ofindividual data points to bout three to ten. The reduction is based uponthe predictive power of the data points when taken as a set.

2. Training—These three to ten data points are then used to trainmultiple machine learning algorithms which then “learn” to recognize, inthis case, patterns of protein masses or gene expression whichdistinguish efficacious drug treatment from non-efficacious. All patientsamples can be used to train the algorithms.

The resulting, trained, algorithms are then tested in order to measuretheir predictive power. Typically, when less than many hundreds oftraining examples are available, some form of cross-validation isperformed. To illustrate, consider a ten-fold cross validation. In thiscase, patient samples are randomly assigned to one of ten bins. In thefirst round of validation the samples in nine of the bins are used fortraining and the remaining samples in the tenth bin are used to test thealgorithm. This is repeated an additional nine times, each time leavingout the samples in a different bin for testing. The results (correctpredictions and errors) from all ten rounds are combined and thepredictive power is then assessed. Different algorithms, as well asdifferent panels, may be compared in this way for this study. The “best”algorithm/panel combination will then be selected. This “smart”algorithm may then be used in future studies to select the patients thatare most likely to respond to treatment.

Many algorithms benefit from additional information taken for thepatients. For example, gender or age could be used to improve predictivepower. Also, data transformations such as normalization and smoothingmay be used to reduce noise. Because of this, a large number ofalgorithms may be trained using many different parameters in order tooptimize the outcome. If predictive patterns exist in the data, it islikely that an optimal, or near-optimal, “smart” algorithm can bedeveloped. If more patient samples become available, the algorithm canbe retrained to take advantage of the new data.

As an example using mass spectrometry, plasma (1 μl) may be applied to ahydrophobic SELDI-target, washed extensively in water, and analyzed bythe SELDI-Tof mass spectrometer. This may be repeated on 100 or morepatient samples. The protein profiles resulting from the intensities ofsome 16,000 m/z values in each sample would be statistically analyzed inorder to identify sets of specific m/z values that are predictive ofdrug efficacy. Identical experiments using other SELDI-targets, such asion-exchange or IMAC surfaces, could also be conducted. These willcapture different subsets of the proteins present in plasma.Furthermore, the plasma may be denatured and prefractionated prior toapplication onto the SELDI target.

Diagnostic & Prognostic Assays

The biomarkers of the present invention can be utilized to determine andtailor treatment therapy in various classes of patients having cancer,including patients who have not received treatment, as well as patientsalready undergoing therapy, such as chemotherapy and radiation. Inaddition, the biomarkers can be used to monitor patients under activetreatment to determine therapeutic efficacy and to indicate whetheradditional chemotherapeutic agents are needed, or whether thetherapeutic course needs to be modified.

Especially useful as a prognostic marker is soluble HER-2 and/or solubleVEGF polypeptide which, when higher than baseline levels are measured inthe blood of subjects, a shorter time to progression of the cancer(e.g., a breast cancer) is observed, especially in those patients whohave received anti-HER-2 therapy. Thus, elevated levels of these markersin patients are indicative of poor prognosis, and suggest the use ofadditional therapy, e.g., sorafenib or other chemotherapeutic agents.

Another useful prognostic marker is soluble EGFR polypeptide which, whenhigher than baseline levels are measured in the blood of subjects, alonger time to death as a result of the cancer (e.g., a breast cancer)is observed, especially in those patients who have received anti-HER-2therapy. Thus, soluble EGFR indicates a more positive prognosis, andpatients with elevated levels of it can benefit from sorafenib (alone orin combination with another chemotherapeutic agent, such as anti-HER-2).

Examples of useful chemotherapeutic agents include, but are not limitedto, e.g., Examples of chemotherapeutic agents include, but are notlimited to, e.g., alkylating agents (e.g., cyclophosphamide, ifosfamide,melphalan, chlorambucil, aziridines, epoxides, alkyl sulfonates),cisplatin and its analogues (e.g., carboplatin, oxaliplatin),antimetabolitites (e.g., methotrexate, 5-fluorouracil, capecitabine,cytarabine, gemcitabine, fludarabine), toposiomerase interactive agents(e.g., camptothecin, irinotecan, topotecan, etoposide, teniposide,doxorubicin, daunorubicin), antimicrotubule agents (e.g., vincaalkaloids, such as vincristine, vinblastine, and vinorelbine; taxanes,such as paclitaxel and docetaxel), inteleukin-2, histone deacetylaseinhibitors, monoclonal antibodies, estrogen modulators (e.g., tamoxifen,toremifene, raloxifene), megestrol, aromatase inhibitors (e.g.,letrozole, anastrozole, exemestane, octreotide), octreotide,anti-androgens (e.g., flutamide, casodex), interferons (e.g.,interferon-alpha, including subtypes thereof, such asinterferon-alpha-2a), etc. See, e.g. Cancer: Principles and Practice ofOncology, 7th Edtion, Devita et al, Lippincott Williams & Wilkins, 2005,Chapters 15, 16, 17, and 63.

The present invention provides methods for determining whether a subjectis at risk for developing a disease or condition characterized byunwanted cell proliferation by detecting biomarkers (e.g., HER-2, EGFR,VEGF, u-PA, p-PAI-1, and soluble forms thereof), that is, nucleic acidsand/or polypeptide markers for cancer.

In clinical applications, human tissue samples may be screened for thepresence and/or absence of biomarkers identified herein. Such samplescould consist of needle biopsy cores, surgical resection samples, lymphnode tissue, or serum. For example, these methods include obtaining abiopsy, which is optionally fractionated by cryostat sectioning toenrich tumor cells to about 80% of the total cell population. In certainembodiments, nucleic acids extracted from these samples may be amplifiedusing techniques well known in the art. The levels of selected markersdetected would be compared with statistically valid groups ofmetastatic, non-metastatic malignant, benign, or normal tissue samples.

In one embodiment, the diagnostic method comprises determining whether asubject has an abnormal mRNA and/or protein level of the biomarkers(e.g., HER-2, EGFR, VEGF, u-PA (urokinase-plasminogen activator),p-PAI-1, and soluble forms thereof), such as by Northern blot analysis,reverse transcription-polymerase chain reaction (RT-PCR), in situhybridization, immunoprecipitation, Western blot hybridization, orimmunohistochemistry. According to the method, cells may be obtainedfrom a subject and the levels of the biomarkers, protein, or mRNA level,are determined and compared to the level of these markers in a healthysubject. An abnormal level of the biomarker polypeptide or mRNA levelsis likely to be indicative of cancer.

Accordingly, in one aspect, the invention provides probes and primersthat are specific to the unique nucleic acid markers disclosed herein.Accordingly, the nucleic acid probes comprise a nucleotide sequence atleast 10 nucleotides in length, preferably at least 15 nucleotides, morepreferably, 25 nucleotides, and most preferably at least 40 nucleotides,and up to all or nearly all of the coding sequence which iscomplementary to a portion of the coding sequence of a marker nucleicacid sequence.

In one embodiment, the method comprises using a nucleic acid probe todetermine the presence of cancerous cells in a tissue from a patient.Specifically, the method comprises:

-   -   1. providing a nucleic acid probe comprising a nucleotide        sequence at least 10 nucleotides in length, preferably at least        15 nucleotides, more preferably, 25 nucleotides, and most        preferably at least 40 nucleotides, and up to all or nearly all        of the coding sequence which is complementary to a portion of        the coding sequence of a nucleic acid sequence and is        differentially expressed in tumors cells;    -   2. obtaining a tissue sample from a patient potentially        comprising cancerous cells;    -   3. providing a second tissue sample containing cells        substantially all of which are non-cancerous;    -   4. contacting the nucleic acid probe under stringent conditions        with RNA of each of said first and second tissue samples (e.g.,        in a Northern blot or in situ hybridization assay); and    -   5. comparing (a) the amount of hybridization of the probe with        RNA of the first tissue sample, with (b) the amount of        hybridization of the probe with RNA of the second tissue sample;        wherein a statistically significant difference in the amount of        hybridization with the RNA of the first tissue sample as        compared to the amount of hybridization with the RNA of the        second tissue sample is indicative of the presence of cancerous        cells in the first tissue sample.

In one aspect, the method comprises in situ hybridization with a probederived from a given marker nucleic acid sequence (e.g., HER-2, EGFR,VEGF, u-PA, p-PAI-1, and soluble forms thereof). The method comprisescontacting the labeled hybridization probe with a sample of a given typeof tissue potentially containing cancerous or pre-cancerous cells aswell as normal cells, and determining whether the probe labels somecells of the given tissue type to a degree significantly different(e.g., by at least a factor of two, or at least a factor of five, or atleast a factor of twenty, or at least a factor of fifty) than the degreeto which it labels other cells of the same tissue type.

Also within the invention is a method of determining the phenotype of atest cell from a given human tissue, for example, whether the cell is(a) normal, or (b) cancerous or precancerous, by contacting the mRNA ofa test cell with a nucleic acid probe at least 12 nucleotides in length,preferably at least 15 nucleotides, more preferably at least 25nucleotides, and most preferably at least 40 nucleotides, and up to allor nearly all of a sequence which is complementary to a portion of thecoding sequence of a nucleic acid sequence, and which is differentiallyexpressed in tumor cells as compared to normal cells of the given tissuetype; and determining the approximate amount of hybridization of theprobe to the mRNA, an amount of hybridization either more or less thanthat seen with the mRNA of a normal cell of that tissue type beingindicative that the test cell is cancerous or pre-cancerous.

Alternatively, the above diagnostic assays may be carried out usingantibodies to detect the protein product encoded by the marker nucleicacid sequence (e.g., HER-2, EGFR, VEGF, u-PA, p-PAI-1, and soluble formsthereof). Accordingly, in one embodiment, the assay would includecontacting the proteins of the test cell with an antibody specific forthe gene product of a nucleic acid, the marker nucleic acid being onewhich is expressed at a given control level in normal cells of the sametissue type as the test cell, and determining the approximate amount ofimmunocomplex formation by the antibody and the proteins of the testcell, wherein a statistically significant difference in the amount ofthe immunocomplex formed with the proteins of a test cell as compared toa normal cell of the same tissue type is an indication that the testcell is cancerous or pre-cancerous. Preferably, the antibody is specificfor HER-2, EGFR, VEGF, u-PA, p-PAI-1, and soluble forms thereof.

The method for producing polyclonal and/or monoclonal antibodies whichspecifically bind to polypeptides useful in the present invention isknown to those of skill in the art and may be found in, for example,Dymecki, et al., (J. Biol. Chem. 267:4815, 1992); Boersma & Van Leeuwen,(J. Neurosci. Methods 51:317, 1994); Green, et al., (Cell 28:477, 1982);and Arnheiter, et al., (Nature 294:278, 1981).

Another such method includes the steps of: providing an antibodyspecific for the gene product of a marker nucleic acid sequence, thegene product being present in cancerous tissue of a Oven tissue type ata level more or less than the level of the gene product in non-canceroustissue of the same tissue type; obtaining from a patient a first sampleof tissue of the given tissue type, which sample potentially includescancerous cells; providing a second sample of tissue of the same tissuetype (which may be from the same patient or from a normal control, e.g.another individual or cultured cells), this second sample containingnormal cells and essentially no cancerous cells; contacting the antibodywith protein (which may be partially purified, in lysed butunfractionated cells, or in situ) of the first and second samples underconditions permitting immunocomplex formation between the antibody andthe marker nucleic acid sequence product present in the samples; andcomparing (a) the amount of immunocomplex formation in the first sample,with (b) the amount of immunocomplex formation in the second sample,wherein a statistically significant difference in the amount ofimmunocomplex formation in the first sample less as compared to theamount of immunocomplex formation in the second sample is indicative ofthe presence of cancerous cells in the first sample of tissue.

The subject invention further provides a method of determining whether acell sample obtained from a subject possesses an abnormal amount ofmarker polypeptide which comprises (a) obtaining a cell sample from thesubject, (b) quantitatively determining the amount of the markerpolypeptide in the sample so obtained, and (c) comparing the amount ofthe marker polypeptide so determined with a known standard, so as tothereby determine whether the cell sample obtained from the subjectpossesses an abnormal amount of the marker polypeptide. Such markerpolypeptides may be detected by immunohistochemical assays, dot-blotassays, ELISA, and the like.

Immunoassays are commonly used to quantitate the levels of proteins incell samples, and many other immunoassay techniques are known in theart. The invention is not limited to a particular assay procedure, andtherefore, is intended to include both homogeneous and heterogeneousprocedures. Exemplary immunoassays which may be conducted according tothe invention include fluorescence polarization immunoassay (FPIA),fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometricinhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA),and radioimmunoassay (RIA). An indicator moiety, or label group, may beattached to the subject antibodies and is selected so as to meet theneeds of various uses of the method which are often dictated by theavailability of assay equipment and compatible immunoassay procedures.General techniques to be used in performing the various immunoassaysnoted above are known to those of ordinary skill in the art.

In another embodiment, the level of the encoded product, oralternatively the level of the polypeptide, in a biological fluid (e.g.,blood or urine) of a patient may be determined as a way of monitoringthe level of expression of the marker nucleic acid sequence in cells ofthat patient. Such a method would include the steps of obtaining asample of a biological fluid from the patient, contacting the sample (orproteins from the sample) with an antibody specific for an encodedmarker polypeptide, and determining the amount of immune complexformation by the antibody, with the amount of immune complex formationbeing indicative of the level of the marker encoded product in thesample. This determination is particularly instructive when compared tothe amount of immune complex formation by the same antibody in a controlsample taken from a normal individual or in one or more samplespreviously or subsequently obtained from the same person.

In another embodiment, the method may be used to determine the amount ofmarker polypeptide present in a cell, which in turn may be correlatedwith progression of a hyperproliferative disorder. The level of themarker polypeptide may be used predictively to evaluate whether a sampleof cells contains cells which are, or are predisposed towards becoming,transformed cells. Moreover, the subject method may be used to assessthe phenotype of cells which are known to be transformed, thephenotyping results being useful in planning a particular therapeuticregimen. For example, very high levels of the marker polypeptide insample cells is a powerful diagnostic and prognostic marker for acancer. The observation of marker polypeptide levels may be utilized indecisions regarding, for example, the use of more aggressive therapies.

As set out above, one aspect of the present invention relates todiagnostic assays for determining, in the context of cells isolated froma patient, if the level of a marker polypeptide is significantly reducedin the sample cells. The term “significantly reduced” refers to a cellphenotype wherein the cell possesses a reduced cellular amount of themarker polypeptide relative to a normal cell of similar tissue origin.For example, a cell may have less than about 50%, 25%, 10%, or 5% of themarker polypeptide compared to that of a normal control cell. Inparticular, the assay evaluates the level of marker polypeptide in thetest cells, and, preferably, compares the measured level with markerpolypeptide detected in at least one control cell, for example, a normalcell and/or a transformed cell of known phenotype.

Of particular importance to the subject invention is the ability toquantitate the level of marker polypeptide as determined by the numberof cells associated with a normal or abnormal marker polypeptide level.The number of cells with a particular marker polypeptide phenotype maythen be correlated with patient prognosis. In one embodiment of theinvention, the marker polypeptide phenotype of a lesion is determined asa percentage of cells in a biopsy which are found to have abnormallyhigh/low levels of the marker polypeptide. Such expression may bedetected by immunohistochemical assays, dot-blot assays, ELISA, and thelike.

Where tissue samples are employed, immunohistochemical staining may beused to determine the number of cells having the marker polypeptidephenotype. For such staining, a multiblock of tissue may be taken fromthe biopsy or other tissue sample and subjected to proteolytichydrolysis, employing such agents as protease K or pepsin. In certainembodiments, it may be desirable to isolate a nuclear fraction from thesample cells and detect the level of the marker polypeptide in thenuclear fraction.

The tissue samples are fixed by treatment with a reagent such asformalin, glutaraldehyde, methanol, or the like. The samples are thenincubated with an antibody, preferably a monoclonal antibody, withbinding specificity for the marker polypeptides. This antibody may beconjugated to a label for subsequent detection of binding. Samples areincubated for a time sufficient for formation of the immunocomplexes.Binding of the antibody is then detected by virtue of a label conjugatedto this antibody. Where the antibody is unlabeled, a second labeledantibody may be employed, for example, which is specific for the isotypeof the anti-marker polypeptide antibody. Examples of labels which may beemployed include radionuclides, fluorescers, chemiluminescers, enzymes,and the like.

Where enzymes are employed, the substrate for the enzyme may be added tothe samples to provide a colored or fluorescent product. Examples ofsuitable enzymes for use in conjugates include horseradish peroxidase,alkaline phosphatase, malate dehydrogenase, and the like. Where notcommercially available, such antibody-enzyme conjugates are readilyproduced by techniques known to those skilled in the art.

In one embodiment, the assay is performed as a dot blot assay. The dotblot assay finds particular application where tissue samples areemployed as it allows determination of the average amount of the markerpolypeptide associated with a single cell by correlating the amount ofmarker polypeptide in a cell-free extract produced from a predeterminednumber of cells.

It is well established in the cancer literature that tumor cells of thesame type (e.g., breast and/or colon tumor cells) may not show uniformlyincreased expression of individual oncogenes or uniformly decreasedexpression of individual tumor suppressor genes. There may also bevarying levels of expression of a given marker gene even between cellsof a given type of cancer, further emphasizing the need for reliance ona battery of tests rather than a single test. Accordingly, in oneaspect, the invention provides for a battery of tests utilizing a numberof probes of the invention, in order to improve the reliability and/oraccuracy of the diagnostic test.

In one embodiment, the present invention also provides a method whereinnucleic acid probes are immobilized on a DNA chip in an organized array.Oligonucleotides may be bound to a solid support by a variety ofprocesses, including lithography. For example, a chip may hold up to250,000 oligonucleotides. These nucleic acid probes comprise anucleotide sequence at least about 12 nucleotides in length, preferablyat least about 15 nucleotides, more preferably at least about 25nucleotides, and most preferably at least about 40 nucleotides, and upto all or nearly all of a sequence which is complementary to a portionof the coding sequence of a marker nucleic acid sequence and isdifferentially expressed in tumor cells. The present invention providessignificant advantages over the available tests for various cancers,because it increases the reliability of the test by providing an arrayof nucleic acid markers on a single chip.

The method includes obtaining a biopsy, which is optionally fractionatedby cryostat sectioning to enrich tumor cells to about 80% of the totalcell population. The DNA or RNA is then extracted, amplified, andanalyzed with a DNA chip to determine the presence of absence of themarker nucleic acid sequences.

In one embodiment, the nucleic acid probes are spotted onto a substratein a two-dimensional matrix or array. Samples of nucleic acids may belabeled and then hybridized to the probes. Double-stranded nucleicacids, comprising the labeled sample nucleic acids bound to probenucleic acids, may be detected once the unbound portion of the sample iswashed away.

The probe nucleic acids may be spotted on substrates including glass,nitrocellulose, etc. The probes can be bound to the substrate by eithercovalent bonds or by non-specific interactions, such as hydrophobicinteractions. The sample nucleic acids can be labeled using radioactivelabels, fluorophores, chromophores, etc.

Techniques for constructing arrays and methods of using these arrays aredescribed, for example, in EP No. 0 799 897; PCT No. WO 97/292 12; PCTNo. WO 97127317; EP No. 0 785 280; PCT No. WO 97/02357; U.S. Pat. No.5,593,839; U.S. Pat. No. 5,578,832; EP No. 0 728 520; U.S. Pat. No.5,599,695; EP No. 0 721 016; U.S. Pat. No. 5,556,752; PCT No. WO95/22058; and U.S. Pat. No. 5,631,734.

Further, arrays may be used to examine differential expression of genesand may be used to determine gene function. For example, arrays ofnucleic acid sequences may be used to determine if any of the nucleicacid sequences are differentially expressed between normal cells andcancer cells. Increased expression of a particular message in a cancercell, which is not observed in a corresponding normal cell, may indicatea cancer-specific protein.

In one embodiment, nucleic acid molecules may be used to generatemicroarrays on a solid surface (e.g., a membrane) such that the arrayednucleic acid molecules may be used to determine if any of the nucleicacids are differentially expressed between normal cells or tissue andcancerous cells or tissue. In one embodiment, the nucleic acid moleculesof the invention may be cDNA or may be used to generate cDNA moleculesto be subsequently amplified by PCR and spotted on nylon membranes. Themembranes may then be reacted with radiolabeled target nucleic acidmolecules obtained from equivalent samples of cancerous and normaltissue or cells. Methods of cDNA generation and microarray preparationare known to those of skill in the art and may be found, for example, inBertucci, et al., (Hum. Mol. Genet. 8:2129, 1999); Nguyen, et al.,(Genomics 29:207, 1995); Zhao, et al., (Gene 156:207); Gress, et al.,(Mammalian Genome 3:609, 1992); Zhumabayeva, et al., (Biotechniques30:158, 2001); and Lennon, et al., (Trends Genet. 7:314, 1991).

In yet another embodiment, the invention contemplates using a panel ofantibodies which are generated against the marker polypeptides of thisinvention. Preferably, the antibodies are generated against HER-2, EGFR,VEGF, u-PA, p-PAI-1, and soluble forms thereof. Such a panel ofantibodies may be used as a reliable diagnostic probe for cancer. Theassay of the present invention comprises contacting a biopsy samplecontaining cells, for example, lung cells, with a panel of antibodies toone or more of the encoded products to determine the presence or absenceof the marker polypeptides.

The diagnostic methods of the subject invention may also be employed asfollow-up to treatment, for example, quantitation of the level of markerpolypeptides may be indicative of the effectiveness of current orpreviously employed cancer therapies as well as the effect of thesetherapies upon patient prognosis.

In addition, the marker nucleic acids or marker polypeptides may beutilized as part or a diagnostic panel for initial detection, follow-upscreening, detection of reoccurrence, and post-treatment monitoring forchemotherapy or surgical treatment.

Accordingly, the present invention makes available diagnostic assays andreagents for detecting gain and/or loss of marker polypeptides from acell in order to aid in the diagnosis and phenotyping of proliferativedisorders arising from, for example, tumorigenic transformation ofcells.

The diagnostic assays described above may be adapted to be used asprognostic assays, as well. Such an application takes advantage of thesensitivity of the assays of the invention to events which take place atcharacteristic stages in the progression of a tumor. For example, agiven marker gene may be up- or down-regulated at a very early stage,perhaps before the cell is irreversibly committed to developing into amalignancy, while another marker gene may be characteristically up- ordown-regulated only at a much later stage. Such a method could involvethe steps of contacting the mRNA of a test cell with a nucleic acidprobe derived from a given marker nucleic acid which is expressed atdifferent characteristic levels in cancerous or precancerous cells atdifferent stages of tumor progression, and determining the approximateamount of hybridization of the probe to the mRNA of the cell, suchamount being an indication of the level of expression of the gene in thecell, and thus an indication of the stage of tumor progression of thecell; alternatively, the assay may be carried out with an antibodyspecific for the gene product of the given marker nucleic acid,contacted with the proteins of the test cell. A battery of such testswill disclose not only the existence and location of a tumor, but alsowill allow the clinician to select the mode of treatment mostappropriate for the tumor, and to predict the likelihood of success ofthat treatment.

The methods of the invention may also be used to follow the clinicalcourse of a tumor. For example, the assay of the invention may beapplied to a tissue sample from a patient; following treatment of thepatient for the cancer, another tissue sample is taken and the testrepeated. Successful treatment will result in either removal of allcells which demonstrate differential expression characteristic of thecancerous or precancerous cells, or a substantial increase in expressionof the gene in those cells, perhaps approaching or even surpassingnormal levels.

In yet another embodiment, the invention provides methods fordetermining whether a subject is at risk for developing a disease, suchas a predisposition to develop cancer, associated with aberrant activityof a polypeptide, preferably, HER-2, EGFR, V EGF, u-PA, p-PAI-1, andsoluble forms thereof, wherein the aberrant activity of the polypeptideis characterized by detecting the presence or absence of a geneticlesion characterized by at least one of (a) an alteration affecting theintegrity of a gene encoding a marker polypeptides, or (b) themis-expression of the encoding nucleic acid. To illustrate, such geneticlesions may be detected by ascertaining the existence of at least one of(i) a deletion of one or more nucleotides from the nucleic acidsequence, (ii) an addition of one or more nucleotides to the nucleicacid sequence, (iii) a substitution of one or more nucleotides of thenucleic acid sequence, (iv) a gross chromosomal rearrangement of thenucleic acid sequence, (v) a gross alteration in the level of amessenger RNA transcript of the nucleic acid sequence, (vi) aberrantmodification of the nucleic acid sequence, such as of the methylationpattern of the genomic DNA, (vii) the presence of a non-wild typesplicing pattern of a messenger RNA transcript of the gene, (viii) anon-wild type level of the marker polypeptide, (ix) allelic loss of thegene, and/or (x) inappropriate post-translational modification of themarker polypeptide.

The present invention provides assay techniques for detecting lesions inthe encoding nucleic acid sequence. These methods include, but are notlimited to, methods involving sequence analysis, Southern blothybridization, restriction enzyme site mapping, and methods involvingdetection of absence of nucleotide pairing between the nucleic acid tobe analyzed and a probe.

Specific diseases or disorders, for example, genetic diseases ordisorders, are associated with specific allelic variants of polymorphicregions of certain genes, which do not necessarily encode a mutatedprotein. Thus, the presence of a specific allelic variant of apolymorphic region of a gene in a subject may render the subjectsusceptible to developing a specific disease or disorder. Polymorphicregions in genes, may be identified, by determining the nucleotidesequence of genes in populations of individuals. If a polymorphic regionis identified, then the link with a specific disease may be determinedby studying specific populations of individuals, for example,individuals which developed a specific disease, such as cancer. Apolymorphic region may be located in any region of a gene, for example,exons, in coding or non-coding regions of exons, introns, and promoterregion.

In an exemplary embodiment, there is provided a nucleic acid compositioncomprising a nucleic acid probe including a region of nucleotidesequence which is capable of hybridizing to a sense or antisensesequence of a gene or naturally occurring mutants thereof, or 5′ or 3′flanking sequences or intronic sequences naturally associated with thesubject genes or naturally occurring mutants thereof. The nucleic acidof a cell is rendered accessible for hybridization, the probe iscontacted with the nucleic acid of the sample, and the hybridization ofthe probe to the sample nucleic acid is detected. Such techniques may beused to detect lesions or allelic variants at either the genomic or mRNAlevel, including deletions, substitutions, etc., as well as to determinemRNA transcript levels.

A preferred detection method is allele specific hybridization usingprobes overlapping the mutation or polymorphic site and having about 5,10, 20, 25, or 30 nucleotides around the mutation or polymorphic region.In a preferred embodiment of the invention, several probes capable ofhybridizing specifically to allelic variants are attached to a solidphase support, for example, a “chip.” Mutation detection analysis usingthese chips comprising oligonucleotides, also termed “DNA probe arrays”is described, for example, by Cronin, et al., (Human Mutation 7:244,1996). In one embodiment, a chip may comprise all the allelic variantsof at least one polymorphic region of a gene. The solid phase support isthen contacted with a test nucleic acid and hybridization to thespecific probes is detected. Accordingly, the identity of numerousallelic variants of one or more genes may be identified in a simplehybridization experiment.

In certain embodiments, detection of the lesion comprises utilizing theprobe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligase chain reaction (LCR) (see, e.g., Landegran,et al., Science 241:1077-1080, 1988; Nakazaw, et al., Proc. Natl. Acad.Sci. USA 91:360-364, 1994), the latter of which can be particularlyuseful for detecting point mutations in the gene (see, e.g., Abravaya,et al., Nuc. Acid Res. 23:675-682, 1995). In an illustrative embodiment,the method includes the steps of (i) collecting a sample of cells from apatient, (ii) isolating nucleic acid (e.g., genomic, mRNA, or both) fromthe cells of the sample, (iii) contacting the nucleic acid sample withone or more primers which specifically hybridize to a nucleic acidsequence under conditions such that hybridization and amplification ofthe nucleic acid (if present) occurs, and (iv) detecting the presence orabsence of an amplification product, or detecting the size of theamplification product and comparing the length to a control sample. Itis anticipated that PCR and/or LCR may be desirable to use as apreliminary amplification step in conjunction with any of the techniquesused for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, et al., Proc. Natl. Acad. Sci. USA 87:1874-1878,1990), transcriptional amplification system (Kwoh, et al., Proc. Natl.Acad. Sci. USA 86:1173-1177, 1989), Q-Beta Replicase (Lizardi, et al.,Bio/Technology 6:1 197, 1988), or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques well known to those of skill in the art. These detectionschemes are especially useful for the detection of nucleic acidmolecules if such molecules are present in very low numbers.

Predictive Assays

Laboratory-based assays, which can predict clinical benefit from a givenanti-cancer agent, will greatly enhance the clinical management ofpatients with cancer. In order to assess this effect, a biomarkerassociated with the anti-cancer agent may be analyzed in a biologicalsample (e.g., tumor sample, plasma) before, during, and followingtreatment.

For example, the expression of HER-2, EGFR, VEGF, u-PA, p-PAI-1, andsoluble forms thereof, mRNA and protein may be detected in plasma. Thus,changes in the baseline plasma concentration of HER-2, EGFR, VEGF, u-PA,p-PAI-1, and soluble forms thereof, may be monitored in patients withcancer. Additionally, HER-2, EGFR, VEGF, u-PA, p-PAI-1, and solubleforms thereof, protein levels may also be monitored by quantitativeimmunohistochemistry using paraffin-embedded tumor biopsies.

Another approach to monitor treatment is an evaluation of serumproteomic spectra. Specifically, plasma samples may be subjected to massspectroscopy (e.g., surface-enhanced laser desorption and ionization)and a proteomic spectra may be generated for each patient. A set ofspectra, derived from analysis of plasma from patients before and duringtreatment, may be analyzed by an iterative searching algorithm, whichcan identify a proteomic pattern that completely discriminates thetreated samples from the untreated samples. The resulting pattern maythen be used to predict the clinical benefit following treatment.

Global gene expression profiling of biological samples (e.g., tumorbiopsy samples, blood samples) and bioinformatics-driven patternidentification may be utilized to predict clinical benefit andsensitivity, as well as development of resistance to an anti-canceragent. For example, RNA isolated from cells derived from whole bloodfrom patients before and during treatment may be used to generate bloodcell gene expression profiles utilizing Affymetrix GeneChip technologyand algorithms. These gene expression profiles may then predict theclinical benefit from treatment with a particular anti-cancer agent.

Analysis of the biochemical composition of urine by 1 D ¹H-NMR (NuclearMagnetic Resonance) may also be utilized as a predictive assay. Patternrecognition techniques may be used to evaluate the metabolic response totreatment with an anti-cancer agent and to correlate this response withclinical endpoints. The biochemical or endogenous metabolites excretedin urine have been well-characterized by proton NMR for normal subjects(Zuppi, et al., Clin Chim Acta 265:85-97, 1997). These metabolites(approximately 30-40) represent the by-products of the major metabolicpathways, such as the citric acid and urea cycles. Drug-, disease-, andgenetic-stimuli have been shown to produce metabolic-specific changes inbaseline urine profiles that are indicative of the timeline andmagnitude of the metabolic response to the stimuli. These analyses aremulti-variant and therefore use pattern recognition techniques toimprove data interpretation. Urinary metabolic profiles may becorrelated with clinical endpoints to determine the clinical benefit.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever.

In the foregoing and in the following examples, all temperatures are setforth uncorrected in degrees Celsius and, all parts and percentages areby weight, unless otherwise indicated.

EXAMPLES

The structures, materials, compositions, and methods described hereinare intended to be representative examples of the invention, and it willbe understood that the scope of the invention is not limited by thescope of the examples. Those skilled in the art will recognize that theinvention may be practiced with variations on the disclosed structures,materials, compositions and methods, and such variations are regarded aswithin the ambit of the invention.

Example 1 Expression Profiling Protocol for Whole Blood Samples

A. Total RNA Isolation from Human Whole Blood

This procedure utilizes the Qiagen QIAamp RNA Blood Mini kit (Version01/99, QIAamp RNA Mini Protocol for Isolation of Total Cellular RNA fromWhole Human Blood).

Tubes of whole blood were stored in a −80° C. freezer. The blood sampleswere partially thaw on ice, that is, thawed until the blood was an icyslurry which will move freely when the tube is inverted. Three volumesof ice-cold Buffer EL was added to one volume of whole blood in a 50-mlconical tube. The samples were mixed by gentle inversion several times,and the samples were placed on ice. The samples were incubated on icefor 10 minutes with gentle inversion during incubation. The samples werethen centrifuged for 10 minutes at 1000 rpm in a tabletop centrifuge at4° C. The supernatant was decanted, and the sample pellets were placedon ice. One volume of ice-cold Buffer EL was added to the samples, andthe samples were gently swirled to resuspend the lymphocyte cell pellet.The samples were transferred to a 15-ml conical tube and placed on ice.An additional 0.5 volume of ice-cold Buffer EL was used to rinse the50-ml tube, and the rinse was added to the samples. The samples werethen centrifuged for 5 minutes at 1000 rpm in a tabletop centrifuge at4° C.

The supernatant was carefully removed, and 580 μl Buffer RLT (containingB-ME) was added to the cell pellets. The samples were then vigorouslyvortexed to solubilize the cell pellets. At this point, the samples maybe stored at in a −80° C. freezer, or the isolation protocol may becontinued with Step 7 of the Qiagen protocol.

The samples were subjected to an on-column DNase digestion using aQiagen RNase-Free DNase Set. The columns were washed both before andafter the digest with 500 μl Buffer RW1, and during the digest, thesamples were incubated for 30 minutes at room temperature. Optional Step12 of the Qiagen protocol was also performed. RNA was eluted withRNase-Free water. Forty-four microliters (44 μl) of RNase-Free water wasadded directly to the filter, allowed to set for I minute, and thencentrifuged for 1 minute at ≧10,000 rpm. The samples of eluted blood RNAwere stored at −80° C. (or the samples may be used immediately).

B. Hybridization of Microarrays

Samples were reverse transcribed to double-stranded cDNA using the GibcoSuperscript II Choice System for RT-PCR according to vendor protocol(Invitrogen, CA).

Samples were organically extracted and ethanol precipitated.Approximately 1 μg cDNA was then used in an in vitro transcriptionreaction incorporating biotinylated nucleotides using an RNA labelingkit (Enzo Diagnostics, NY). The resulting cRNA was put through an RNeasyclean-up protocol and then quantified using UV spectrophotometry. ThecRNA (15 μg) was fragmented in the presence of MgOAc and KOAc at 94° C.Fragmented RNA (10 μg) wa loaded onto each array, one cRNA sample perarray. Arrays were hybridized for 16 hours at 45° C. rotating at 60 rpmin an Affymetrix GeneChip Hybridization Oven 640.

C. Data Analysis

Following hybridization, arrays were stained withPhycoerythrin-conjugated Streptavidin, placed in an Agilent GeneArrayScanner and then exposed to a 488 nm laser, causing excitation of thephycoerythrin. The Microarray Suite 5.0 software digitally converts theintensity of light given off by the array into a numeric valueindicative of levels of gene expression.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention and, withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

1.-3. (canceled)
 4. A method for treating a patient with sorafenib forbreast cancer after initial therapeutic treatments, based on thepatient's response during the course of therapeutic treatment, saidmethod comprising treating the patient with the same amount ofsorafenib, a different amount of sorafenib, adding an additionaltherapeutic treatment or ceasing treatment with sorafenib, in view ofthe effectiveness of the treatment with sorafenib as determined bycomparing the level of expression of one or more biomarker(s) selectedfrom the group consisting of HER-2, EGFR, and soluble forms thereof, inat least a second biological sample with the level of expression of theone or more biomarker(s) in a first biological sample; wherein (a) thefirst biological sample is taken from the patient prior to treatmentwith sorafenib; and (b) the at least second biological sample is takenfrom the patient subsequent to the initial treatment with sorafenib; andwherein a change in the level of expression of the one or morebiomarker(s) in the second biological sample compared to the level ofexpression of the one or more biomarker(s) in the first biologicalsample as compared to statistically valid changes in the baseline levelof expression of the biomarker(s) in other patients showing an effectiveresponse to sorafenib indicates the effectiveness or lack thereof of thetreatment with sorafenib, and wherein higher levels of expression ofHER-2 or a soluble form thereof indicates a poorer response and/orprognosis, and higher levels of expression of EGFR or a soluble formthereof indicates a better response and/or prognosis.
 5. A method oftherapeutic treatment with sorafenib for a subject having a breastcancer and having a high baseline level of soluble EGFR, said methodcomprising administering further treatment based on a prediction oftherapeutic outcome; wherein said prediction is based on a statisticallyvalid comparison of the change in the level of expression of solubleEGFR in said subject in response to prior treatment with sorafenib, withchanges in levels of expression from the subject's baseline of solubleEGFR, to the change in level of expression of soluble EGFR before andafter treatment with sorafenib in other breast cancer patients having ahigh baseline level of soluble EGFR and having a clinically beneficialresponse to sorafenib, to predict the clinical benefit of continuing theadministration of effective amounts of sorafenib; wherein changes inlevels of expression of soluble EGFR are determined by: (a) measuringthe level of expression of soluble EGFR in said subject afteradministration of sorafenib, (b) determining the change in the level ofexpression of soluble EGFR in said subject after administration ofsorafenib, from the high baseline levels of soluble EGFR; whereinstatistically valid changes from baseline expression levels ofpreviously treated patients having a clinically beneficial response areused to predict clinical benefit of further treatment, wherein saidfurther treatment comprises administering to the subject the same amountof sorafenib, a different amount of sorafenib, adding an additionaltherapeutic treatment or ceasing treatment with sorafenib, and whereinhigher levels of expression of soluble EGFR indicate a better responseand/or prognosis.